Field of the Invention
The subject of the invention is a method and apparatus for converting components of a condensed phase sample into gaseous ions and analysis thereof.
During the implementation of the method according to the invention a high-velocity liquid jet is impacted with the surface of said sample. The liquid droplets formed at the impact of the liquid with the surface of the sample carry away the components of the sample (desorption step). The sample remaining after the evaporation of the solvent is a gaseous ion itself or it is convertible into gaseous ion by the use of an external effect—heat, electromagnetic effect etc. (optional step for providing gaseous ions). The obtained gaseous ions are analyzed, preferably by mass spectrometry or ion mobility spectrometry (detection step).
Description of the Related Art
Mass spectrometric ionization methods have been traditionally developed for the analysis of gaseous or volatile materials. Disadvantage of these ionization methods is that they lack the capability of analysis of non-volatile compounds. This group of compounds includes e.g. peptides, proteins, nucleic acids, carbohydrates. From the 1970's a new family of ionization methods has been developed which was able to convert condensed phase molecules directly into ions on the gas/solid interface and subsequently transfer the nascent ions to the gas phase. These ionization methods are generally named as “desorption ionization” methods since they are coupled with the desorption of the formed ions. First desorption ionization method was the so-called field desorption ionization generated by electric field, which utilizes the high electric field gradient formed around the edges of sharp surface features for the parallel ionization and desorption of the molecules present on surface. [Beckey, H. D., Organic Mass Spectrometry 6 (6), 6558-(1972)]. The disadvantage of this method is that the sample has to be deposited onto the very edge of emitter needles and the geometry of emitter tips has a strong influence on ionization efficiency.
The next generation of desorption ionization methods are based on an alternative way of ionization by utilizing a so-called analytical beam for ionization. In this method a beam comprising high energy ions, atoms or photons is impacted with the surface of the studied sample. Impact of analytical beam on surface produces some gaseous ions and molecules deriving from the studied surface. First method utilizing analytical beam was plasma desorption ionization which employed high energy particles produced by radioactive decay [Macfarlane, R. D. et al., Science, 191 (4230), 920-925 (1976)].
While in case of plasma desorption a poorly defined beam was utilized, secondary ion mass spectrometry (SIMS), which was developed practically at the same time, employed a well-defined analytical beam of ions accelerated by electric potential difference [Bennighoven, A., Surface Science 28(2) 541-(1971)]. SIMS provides an excellent spatial resolution, due to the small diameter of ion beams, but the molecular weight range of molecules, which undergo SIMS ionization, is limited. Method can also be used for in-depth analysis, however, in this case the molecular weight limits are more critical since the formed ions contain only 1 or 2 atoms. Study of liquid samples was developed first time in the case of SIMS ionization [liquid SIMS; LSIMS, Aberth, W., Analytical Chemistry, 54 (12): 2029-2034 (1982)]. LSIMS has softer range limits compared to the original method, e.g. small protein molecules can be ionized by it. Disadvantage of LSIMS is that the samples have to be dissolved in a solvent having high surface tension and low vapour pressure such as glycerol. This step often involves solubility problems, and dissolution of solid samples obviously excludes the possibility to obtain information about spatial distribution of the molecules of a sample.
A further developed version of LSIMS is the “fast atom bombardment” (FAB) method [Williams, D. H. et al., JACS, 103 (19): 5700-5704 (1981)]. The technique is based on the electrostatic acceleration of noble gas ions followed by neutralization, yielding a neutral beam of nobel gas atoms having yet a high energy level which can be utilized for ionization. FAB ionization is also suitable for the analysis of liquid phase samples.
Another direction of development of SIMS technique has led to the so-called massive cluster impact (MCI) ionization [Massive cluster impact; MCI, Mahoney, J. F., Rapid Communications in Mass Spectrometry, 5 (10): 441-445 (1991)] which utilizes multiply charged glycerol clusters instead of the traditionally applied gold ions. This technique can be applied for the analysis of solid surfaces and the weight of the analyzed molecules is not limited practically. A further advantage of this technique is, compared to SIMS, that multiply charged ions are formed which can be analyzed more effectively by mass spectrometry.
Common disadvantage of described methods is that all of them work strictly under high vacuum conditions. Hence, samples are introduced into high vacuum regime of mass spectrometers, which involves strong restrictions on the composition and size of samples.
Laser desorption ionization methods, where laser was applied as analytical beam, have been developed from the early 1980's [Cooks, R. G. et al., JACS, 103 (5): 1295-1297 (1981)]. Simple laser desorption ionization, similarly to SIMS, gives poor ionization efficiencies and they can only be used for the study of a very limited scope of molecules. Application area of laser desorption methods was dramatically extended by the application of so-called matrix compounds. Matrix compounds, which are present in great excess, are generally mixed to sample in solution phase and the mixture is co-crystallized onto solid carrier and the obtained crystallized sample is analyzed by means of laser desorption, i.e. where laser is used as an analytical beam. The new method was named as matrix-assisted laser desorption ionization (MALDI) [Karas, Hillenkamp, Analytical Chemistry, 60 (20): 2299-2301 (1988)]. The technique can be applied generally for the analysis of macromolecular compounds such as polymers, proteins, carbohydrates and nucleic acids. Main disadvantage of MALDI is that the technique requires embedding of analyte molecules into matrix crystal lattice, thus analysis of natural surfaces is problematic.
Need for desorption ionization methods working under atmospheric conditions has been raised recently. Advantages of atmospheric pressure desorption ionization method include: (1) samples are not introduced into vacuum regime of mass spectrometer, which makes the analytical procedure faster, (2) since sample does not enter vacuum, there is no need for the removal of volatile components, (3) arbitrary objects can be investigated this way, (4) living organisms can be studied directly. Desorption ionization methods utilizing high-velocity beam of atoms or ions cannot be used under atmospheric pressure conditions, since particles cannot be accelerated to suitable velocities at high pressure due to consecutive collisions with gas molecules and this phenomenon is responsible for the divergence of particle beams.
Among the above described methods, only the MALDI ionization can be implemented at atmospheric pressure without changes in instrumentation since laser beams do not interact with air molecules. Atmospheric pressure MALDI was developed by Laiko et al. in 2002. However, the technique did not spread widely due to low ion yield which is further decreased by the substantial ion loss in the atmospheric interface, and workplace safety issues generally associated with the use of laser in open experimental setups.
The recently developed desorption electrospray ionization (DESI; Takats et al., Science, 306 (5695): 471-471, 2004) is practically the atmospheric pressure version of MCI technique described above, but having the important difference that the droplets are produced by electrospray method and accelerated to the desired velocity by supersonic gas stream instead of electrostatic field gradient. Nevertheless, DESI has fulfilled all expectations associated with atmospheric pressure desorption ionization methods, so it opened the door to the mass spectrometric analysis of arbitrary objects independently from their chemical composition and size. In the course of DESI process, high-velocity electrosprayed droplets impact with sample surface. Impacting droplets dissolve molecules present on surface and emit secondary droplets which are also charged. These charged secondary droplets produce ions finally via complete evaporation of solvent.
Although DESI technology bears a number of advantages compared to previously developed desorption methods, it also shows drawbacks on several fields of applications. At first, DESI is a strict surface analysis method, thus it lacks the capability of in-depth analysis in case of most samples. At second, since the atmospheric pressure beam of charged droplets is intrinsically divergent due to Coulomb repulsion among charged particles, it hinders the high-resolution chemical imaging. Furthermore, although DESI is fully compatible with biological tissues from analytical point of view, in vivo application of DESI was shown to cause embolism in various animal models. Embolism was tentatively associated with the supersonic nitrogen stream applied for the acceleration of the droplets, which pumps gas bubbles into the tissue and causes embolism.
In order to provide solution for the above listed problems, a need has emerged for an alternative atmospheric pressure desorption ionization method which employs collimated and high-energy analytical beam where the beam is not constituted by droplets accelerated by high-velocity gas. The aim of our research was to develop an atmospheric pressure ionization method which is capable of in-depth analysis of samples, high-resolution chemical imaging and in-vivo analysis.